Effection Of IL-1β On Schwann Cells In Peripheral Nervous System Wallerian Degeneration

BackgroundThe ultimate goal of regenerative medicine research is to enable replacement of lost or damaged tissues or organs. Regeneration can potentially be accomplished using the processes of de-differentiation, trans-differentiation or reprogramming. Humans have a limited capacity to regenerate tissues or organs, including liver and the peripheral nervous system. In some cases, the process of regeneration involves de-differentiation of mature cells. De-differentiation is a mechanism in which terminally-differentiated cells revert to a less-differentiated stage within the same lineage and allows cells to proliferate before re-differentiating, leading to the replacement of lost cells.Schwann cells, myelinated glial cells of the peripheral nervous system, de-differentiate and revert to an immature phenotype in Wallerian degeneration following nerve injury and, by doing so, can actively promote nerve repair and functional recovery. Following de-differentiation, Schwann cells contribute to macrophage-mediated myelin removal and re-enter the cell cycle, proliferate, and then form bands of Bungner, which support and direct outgrowing axons to sites of innervation. Moreover, these cells express and secrete axonal growth promoting factors, then re-differentiate and myelinate regenerated axons, which eventually leads to substantial functional recovery. This sequence emphasizes the central function of Schwann cell de-differentiation in peripheral nervous system regeneration.Wallerian degeneration can be consider an innate-immune response of the peripheral nervous system to traumatic nerve injury. Peripheral nervous system injury induces immune and non-immune cells to produce cytokines and develop an efficient cytokine network during Wallerian degeneration. Before macrophage recruitment, injured peripheral nervous system product IL-1β and other inflammatory cytokines. Certain inflammatory cytokines can influence de-differentiation in some types of terminally differentiated cells. For example, IL-1β induces chondrocyte de-differentiation.Before and after macrophage recruitment, Wallerian degeneration can be defined as two phases characterized by cytokine protein production profiles. The first phase is characterized by the synthesis of IL-1βand other inflammatory cytokines (such as TNF-α, IL-la, GM-CSF and IL-6). The second phase is characterized by the production of 11-10,11-6, and a GM-CSF inhibitor molecule, and furthermore, by the diminished production of IL-1β.Therefore, the first phase is largely inflammatory and the second is predominantly anti-inflammatory. Thus, based on our research and previous reports, we interpret that appropriate concentrations of IL-1βare conductive to de-differentiation and regeneration of Schwann cells during the first phase of Wallerian degeneration.The transcription factor c-JUN is a key regulator of Wallerian degeneration, governs major aspects of injury response, determines the expression of trophic factors, adhesion molecules, the formation of regeneration tracks and myelin clearance and controls the distinctive regenerative potential of peripheral nerves. A key function of c-JUN is the activation of a repair program in Schwann cells and the creation of a cell specialized to support regeneration. Among potential intracellular activators of c-JUN is the AP-1 transcription complex, of which c-JUN is a key component. The c-JUN/AP-1 complex is a prominent downstream nuclear target of ERK and JNK pathways.We hypothesized that, in addition to a role in immune responses, IL-1β participates in Schwann cells de-differentiation, proliferation and apoptosis. We used a rodent in in-vitro Wallerian degeneration model to investigate effects of IL-1β on Schwann cell de-differentiation, proliferation and apoptosis, and to investigate potential mechanisms involved in the effects, excluding effects of immune cells and other inflammatory cytokines.ObjectiveTo investigate effects of IL-1β on Schwann cell de-differentiation, proliferation and apoptosis, and to investigate potential mechanisms involved in the effects, we used a rodent in in-vitro Wallerian degeneration model treated with IL-1β excluding effects of immune cells and other inflammatory cytokines.Methods1. To establish in-vitro Wallerian degeneration model. Sciatic nerve explant cultures were performed as previously reported by Thomson et al. (1993) with minor modifications. Sciatic nerve explants without epineurium were established by loosely separating small bundles of fibers from the isolated nerve. The bundles of nerve were maintained in DMEM containing 10% fetal bovine serum,100 U/ml penicillin,100 mg/ml streptomycin and 0.25 mg/ml amphotericin B at 37℃ with 5% CO2. Nerve bundles were treated with or without various concentrations of recombinant rat IL-1β. Schwann cells and fibroblasts compose most of the non-neuronal cell population in intact peripheral nervous system, whereas macrophages, which are scarce in intact peripheral nervous system, are recruited in large numbers from the third day after injury. In our modified in-vitro Wallerian degeneration model, in which epineurium were carefully detached, Schwann cells comprise the majority of the cell population in this model and are the primary object of this study.2. Real time PCR was used to detect relative quantification of Il-1β mRNA in Schwann cells harvested from in-vitro Wallerian degeneration model at different times(6 H,12 H,24 H).3. Elisa was used to detect relative quantification of IL-1βprotein in Schwann cells harvested from in-vitro Wallerian degeneration model at different times(12 H,24 H,36H,48H).4. Immunofluorescent staining was used to demonstrate expression of p75NTR, a marker of Schwann cell de-differentiation, and MPZ, an essential factor in myelination, in Schwann cells of in-vitro Wallerian degeneration model treated with various concentrations of recombinant rat IL-1β(0,5, and 50 ng/ml) and harvested after 48 h. The expression of p75NTR and MPZ were quantitatively verified by Western blotting.5. The expression of p75ntr and Mpz mRNAs were quantitatively verified by Real time PCR in Schwann cells harvested from in-vitro Wallerian degeneration model treated with or without 5 ng/ml IL-1βat 6,12 and 24 h.6. We used immunofluorescent staining to detect the expression of c-JUN in Schwann cell nuclei. Schwann cells harvested from in-vitro Wallerian degeneration model treated with or without 5 ng/ml IL-1β after 24 h. The percent analysis of endonuclear c-JUN(+) cells was quantitatively verified. And the expression of c-JUN were quantitatively verified by Western blotting.7. The expression of c-Jun mRNAs were quantitatively verified by Real time PCR in Schwann cells harvested from in-vitro Wallerian degeneration model treated with or without 5 ng/ml IL-1βat 6,12 and 24 h.8. The AP-1 activity assay was used to examine AP-1 activity of Schwann cells in the in-vitro Wallerian degeneration model with or without 5 ng/ml IL-1β at 6,12, and 24 h.9. We used immunofluorescent staining to detect the expression of Ki67 in Schwann cell nuclei. Schwann cells harvested from in-vitro Wallerian degeneration model treated with or without 5 ng/ml IL-1βafter 48 h. The percent analysis of endonuclear Ki67(+) cells was quantitatively verified.10. Tunel was used to detect apoptosis of Schwann cells harvested from in-vitro Wallerian degeneration model treated with or without 5 ng/ml IL-1βafter 48 h. The percent analysis of apoptosis cells was quantitatively verified.11. Western blotting was used to detect expression of Bcl-2 and Bax of Schwann cells harvested from in-vitro Wallerian degeneration model treated with or without 5 ng/ml IL-1βafter 24 h.12. Data are expressed as the mean ±SE. The significance of differences between two independent samples was statistically assessed using Student’s t-test. The statistical significance of differences between groups was determined by one-way ANOVA followed by the Least Significant Difference (LSD) test. P-values< 0.05 were considered significant.Results1. Relative quantification of Il-1β mRNA harvested from Schwann cells of in-vitro Wallerian degeneration model at different times(6 H,12 H,24 H)was performed by Real time PCR, and results were analyzed by one-way ANOVA followed by LSD test. The analysis revealed expression of Il-1β mRNA by Schwann cells increased after nerve damage, and was elevated approximately seven-fold at 12 h.2. Relative quantification of IL-1β protein harvested from Schwann cells of in-vitro Wallerian degeneration model at different times(12 H,24 H,36H,48H) was performed by Elisa, and results were analyzed by one-way ANOVA followed by LSD test. The analysis revealed expression of IL-1β protein by Schwann cells increased after nerve damage, and was increased five-fold at 36 h.3. I mmunofluorescent staining of p75NTR and MPZ, and western blotting to quantitatively analyze expression levels in Schwann cells harvested from an in-vitro Wallerian degeneration model treated with various concentrations of IL-1β(0,5, and 50 ng/ml) at 48 h. The results of western blotting were analyzed by one-way ANOVA followed by the LSD test. Sciatic nerve Schwann cells were treated with various concentrations of recombinant rat IL-1β (0,5, and 50 ng/ml) in this in-vitro Wallerian degeneration model and harvested after 48 h. We detected expression of p75NTR, a marker of Schwann cell de-differentiation, and MPZ, an essential factor in myelination in these Sciatic nerves to assess Schwann cells de-differentiation. Immunofluorescent staining demonstrated that expression of p75NTR increased and levels of MPZ decreased in the 5 ng/ml IL-1β group compared with 0 and 50 ng/ml groups at 48 h. Additionally, differences in p75NTR and MPZ expression between the 50 and 0 ng/ml IL-1β groups were not obvious. These were quantitatively verified by Western blotting. The analysis revealed expression of p75NTR by Schwann cells increased by about 70% in the 5 ng/ml group in comparison to the 0 ng/ml group, and expression of MPZ also decreased by 30%. In addition, Western blotting demonstrated that the differences in p75NTR and MPZ expression between the 50 and 0 ng/ml IL-1β groups were not statistically significant. These results suggested that 5 ng/ml IL-1β promoted Schwann cells de-differentiation. Nevertheless, a high concentration (50 ng/ml) IL-1βdid have similar effects.4. Schwann cells were harvested from in-vitro Wallerian degeneration model with or without 5 ng/ml IL-1βat 6,12 and 24 h. Real time PCR demonstrated that expression of p75ntr mRNA increased in a time-dependent manner in each concentration group, and was elevated more significantly in the 5 ng/ml group compared with the 0 ng/ml group, specifically, elevated five-fold at 6 h and approximately 2.5 times at 12 and 24 h. Additionally, expression of Mpz mRNA decreased in a temporal manner in both concentration groups, and further decreased in the 5 ng/ml group compared with the 0 ng/ml group, which were all decreased by about 50% at different times. These results suggested that 5 ng/ml IL-1βincreased expression of the de-differentiation gene p75ntr and decreased expression of the myelination locus Mpz, promoting Schwann cells de-differentiation.5. Immunofluorescent staining was used to detect the expression of c-JUN in Schwann cell nuclei. Schwann cells harvested from in-vitro Wallerian degeneration model treated with or without 5 ng/ml IL-1βafter 24 h. Immunofluorescent staining demonstrated that levels of c-JUN in Schwann cell nuclei increased in the 5 ng/ml group compared with the 0 ng/ml group. This was quantitatively verified by percent analysis of endonuclear c-JUN(+) cells, and the percent was increased by approximately 35% in the 5 ng/ml group compared with the 0 ng/ml group at 24 h. Western blot analysis also revealed that expression of c-JUN in Schwann cells was significantly increased by about 75% in the 5 ng/ml group in comparison to the 0 ng/ml group at 24 h.6. We used Real time PCR to detect expression of c-Jun mRNA in Schwann cells in our in-vitro Wallerian degeneration model with or without 5 ng/ml IL-1βat 6,12, and 24 h. Real time PCR demonstrated that expression of c-Jun mRNA increased in a time-dependent manner in both concentration groups, and increased more significantly at 24 h (elevated by about 48%) in the 5 ng/ml group compared with the 0 ng/ml group. These results suggested that 5 ng/ml IL-1βincreased expression of c-Jun and c-JUN transcription factor, and elevated the proportion of endonuclear c-JUN(+) cells in Schwann cells.7. We examined AP-1 activity of Schwann cells in the in-vitro Wallerian degeneration model with or without 5 ng/ml IL-1βat 6,12, and 24 h using an AP-1 activity assay. This indicated that AP-1 activity increased in a time-dependent manner in both concentration groups, and was more significantly increased (by about 56% and 82%, respectively) at 12 and 24 h in the 5 ng/ml group compared with the non-exposed group. AP-1 activity assay results, combined with analyses of c-JUN, suggested that 5 ng/ml IL-1β promoted endonuclear c-JUN expression and stimulation of AP-1 activity in our in-vitro Wallerian degeneration model.8. Immunofluorescent staining was used to detect the expression of Ki67 in Schwann cell nuclei. Schwann cells harvested from in-vitro Wallerian degeneration model treated with or without 5 ng/ml IL-1βafter 48 h. Immunofluorescent staining demonstrated that levels of Ki67 in Schwann cell nuclei increased in the 5 ng/ml group compared with the 0 ng/ml group. This was quantitatively verified by percent analysis of endonuclear Ki67(+) cells, and the percent was increased in the 5 ng/ml group compared with the 0 ng/ml group at 48 h.9. Tunel was used to detect the percentage of Schwann cells apoptosis. Schwann cells harvested from in-vitro Wallerian degeneration model treated with or without 5 ng/ml IL-1βafter 48 h. Tunel demonstrated that the percentage of Schwann cells apoptosis decreased in the 5 ng/ml group compared with the 0 ng/ml group. Western Blot was used to detect the expression of Bcl-2 and Bax in Schwann cells harvested from in-vitro Wallerian degeneration model treated with or without 5 ng/ml IL-1βafter 24 h. Western Blot demonstrated that the expression of Bcl-2 increased and the expression of Bax decreased in the 5 ng/ml group compared with the 0 ng/ml group. In addition, the ration of Bcl-2/Bax increased in the 5 ng/ml group compared with the 0 ng/ml group.ConclusionOur study demonstrates that in-vitro Wallerian degeneration model is an effective model to investigate effects of IL-1β on Schwann cell de-differentiation in peripheral nervous system Wallerian degeneration excluding effects of immune cells and other inflammatory cytokines. We used the rodent in-vitro Wallerian degeneration model to demonstrates that expression of Il-1β mRNA and IL-1β protein by Schwann cells increase after nerve damage and Wallerian degeneration can be consider an innate-immune response of the peripheral nervous system to traumatic nerve injury.Schwann cells of Wallerian degeneration produce IL-1β which promotes Schwann cells de-differentiation and regeneration via the c-JUN/AP-1 signaling pathway. The precise molecular mechanisms of IL-1βregulation of c-JUN/AP-1 activity are not fully understood, and further studies are required. In addition, Schwann cells of Wallerian degeneration produce IL-1βwhich promotes Schwann cells proliferation, which may be also via the c-JUN/AP-1 signaling pathway. And IL-1βinduces inhibition of Schwann cells apoptosis via the Bcl-2/Bax signaling pathway.In a word, Schwann cells of Wallerian degeneration produce IL-1β which promotes Schwann cells de-differentiation and regeneration via the c-JUN/AP-1 and other signaling pathway.